New strategy for cancer therapy using Hi-resolution microsatellite instability analysis and Real-time quantitative reverse transcriptase polymerase reaction

微转移：用实时定量逆转录聚合酶链式反应(RT-PCR)检测骨髓和外周血中的播散性癌细胞。我们比较了CEA、CK18、CK19和CK20在20个细胞系中的表达，以选择最合适的。对13例健康志愿者外周静脉血中CK-18mRNA的表达进行了分析，发现CK-18mRNA在肿瘤细胞系中的表达明显低于正常对照。然而，所有样本的表达水平都达到了常规RT-PCR检测到的水平。因此，不仅要对靶基因进行定性，而且要对靶基因进行定量分析，以检测微转移。定量RT-PCR方法将对目标基因表达水平的可能差异进行比较。对于临床应用，还需要更多的研究。微卫星不稳定性：我们使用高分辨率荧光微卫星分析(HFRMA)，研究了800多名散发性乳腺癌、胃癌、结肠癌、肺癌和肝癌患者。在该系统中，为了提高聚合酶链式反应产物的重现性、电泳迁移的准确性和检测系统的特性，我们准备了几个装置。在该系统中观察到的所有微卫星变化可分为两类：A类微卫星序列变化较小，在有限的座位上观察到；B类微卫星序列变化剧烈，分布广泛。前者被认为与DNA错配修复(MMR)的异常活动有关。

Micrometastasis; Real time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect disseminated cancer cells in bone marrow and peripheral blood. We compared CEA, CK18, CK19 and CK20 expression using 20 of cell lines in order to select the most appropriate. In an analysis of CK 18 mRNA expression in peripheral venous blood in 13 healthy volunteers, we found that CK 18 mRNA was much less expressed than in cancer cell lines. However, the expression in all samples was at a level which was also detected using conventional RT-PCR. It would thus seem that not only qualitative, but also quantitative analysis, of the target mRNA is important to detect micrometastasis. Quantitative RT-PCR methods will make comparisons of the possible differences in expression levels of the target gene. For clinical applications, much further study is needed.Microsalellite Instability; Using high-resolution fluorescent microsatellite analysis (HFRMA), we studied over 800 patients with sporadic breast, gastric, colon, lung and liver cancer. In this system, several devices were prepared to improve reproducibility of polymerase chain reaction products, migration accuracy of electrophoresis, and characteristics of the detection system. All the microsatellite changes observed in this system can be classified into two types: type A with relatively small changes in microsatellite sequences observed in limited loci and type B with drastic and widely dispersed changes. The former was thought to be connected to abnormal activity in DNA mismatch repair (MMR).